(i) Field of Invention
The present invention relates to the field of molecular biology and recombinant DNA-technology (genetic engineering). The invention allows a simplified in vitro synthesis of nucleic acids. The herein described methods are especially applicable to the amplification of nucleic acids with primer-dependent DNA/RNA-polymerases, DNA- and RNA-ligases. The invention has numerous applications in molecular biology, medical diagnostics, environmental and forensic analysis.
The invention relates especially to a process for isothermal, non-transcription based, amplification of nucleic acids and nucleic acid sequences respectively, by means of enzymes whereby the nucleic acids and nucleic acid sequences respectively, may be at least partially separated into single strands and/or transcribed.
The amplification proceeds by enzymatic incorporation of at least two oligonucleotide building blocks with a sequence essentially complementary to the sequence of the ends of the complementary strands of the nucleic acid or nucleic acid sequence to be amplified, wherein the product of the building blocks itself corresponds to the nucleic acid and nucleic acid sequence to be amplified.
(ii) Description of Related Art
The in vitro-amplification of nucleic acids, such as DNA and RNA and nucleic acid sequences, respectively, has many applications and may be performed in different ways (amplification in this document means a substantial amplification, i.e. more than doubling of the starting material).
The principle of amplification of DNA by means of polymerase activity has been described in detail by Kleppe et al., J. Mol. Biol. 56: 341 (1971). Accordingly, oligonucleotides are used as starting points for enzymatic DNA-synthesis such that the product of this synthesis may be used as template for further synthesis. According to this principle the complementary nucleic acids strands have to be separated repeatedly between the single steps of the synthesis. U.S. Pat. No. 4,683,202 describes the realization of this principle.
Regarding the applied temperature one may distinguish especially two processes: processes with cycling temperature changes between amplification temperature and denaturing (strand separation) temperature, wherein the nucleic acids or the nucleic acid sequences prior to the amplification step have to be separated essentially completely into single strands. The other amplification processes work isothermally.
The polymerase chain reaction (PCR), for example, belongs to the first group of temperature cycling processes, wherein a nucleic acid sequence can be amplified exponentially in such a reaction where the temperature is subjected to a cyclic change. For such an amplification, usually a thermostable polymerase is used [e.g. Saiki et al., Science, 230, 1350-1354 (1985), Saiki et al., Science, 239, 487 (1988), respectively].
According to U.S. Pat. No. 4,683,195 (Mullis et al.) one can perform in a temperature cycling process the strand separation following the amplification step by any suitable denaturation method, it may be chemically, physically or enzymatically. In the referred document physical strand separation methods are preferred, e.g. heating the nucleic acid until complete denaturation (&gt;99%), i.e. separated into two single strands. A typical heat denaturation is performed at temperatures between 90.degree. C. and 105.degree. C. and lasts generally between 0.5 and 3 minutes. Preferred temperatures are between 90.degree. C. and 100.degree. C. for 0.5 to 3 minutes. When using a thermolabile enzyme (U.S. Pat. No. 4,683,202) one has to add fresh enzyme after each strand separation by heat. By using a thermostable enzyme one does not need to interrupt the thermocycling for enzyme addition. The process is performed by means of temperature cycling apparatus "simultaneously".
A further process in the group of thermocycling processes is the so called ligase chain reaction (LCR) e.g. according to F. Barany in Proc. Natl. Acad. Sci. USA, 88, 189-193 (1991), wherein the process, as in PCR, leads to a non-linear amplification of the starting nucleic acid, also a cyclic temperature change proceeds between an amplification temperature and a denaturation temperature and a thermostable ligase is used as enzyme (WO 90/01069 and EP-A 320308). A special form of LCR is the "gap-filling LCR", in which process, in addition to the ligase also a polymerase is used. In such a reaction only the products of the polymerase reaction are used a substrates for the ligase (U.S. Pat. No. 5,427,930).
One may also use several similar enzymes with slightly different properties in one reation to achieve special results, which cannot be achieved with only one enzyme. The amplification of long DNA-stretches is an example for such a reaction (Barnes, W. M., Proc. Natl. Acad. Sci. USA, 91, 2216-2220, (1994)).
On the other hand, isothermal processes, such as e.g. the self sustained sequence reaction (3SR) are well known in the state of the art, e.g. from E. Fahy et al., PCR Meth. Appl. 1, 25-33, (1991); D. Kwoh et al., Proc. Natl. Acad. Sci. (USA) 86, 1173-7 (1989); WO 88/10315 (T. R. Gingeras et al.); EP-A 0 329 822 (H. I. Miller et al.); and J. van Brunt, Bio/Technology 8, 291-4(1990); U.S. Pat. Nos. 5,409,818, 5,399,491 and 5,194,370).
These processes have in common that they can be performed isothermally, however, one needs several different enzymes for the amplification (e.g. reverse transcriptase, RNaseH, RNA-polymerase) as the amplification step is performed via transcription (RNA to DNA, and DNA to RNA, respectively). A further disadvantage is also the lower specificity of such isothermal processes of the state of the art as there are no thermostable enzymes used. If RNA-products are the result, a further disadvantage is that handling is more difficult (size fractionation is not as simple as with DNA). There is also no contamination control system available.
The following references describe further alternative processes for the amplification of nucleic acids and nucleic acid sequences, respectively, by means of specific enzyme systems (restriction endonucleases, Q-beta-replicases): G. T. Walker et al., Proc. Natl. Acad. Sci. (US) 89, 392-6 (1992); U.S. Pat. No. 5,356,774 (V. D. Axelrod et al.); and P. Knight, Bio/Technology 7, 609-10 (1989).
WO 90/10064 and WO 91/03573 describe the use of the origin of replication of phage phi29 for the isothermal amplification of nucleic acids.
The so-called strand displacement amplification (U.S. Pat. Nos. 5,455,166 and 5,422,252) is based on the strand displacement activity of polymerases, which start to displace one of the complementary nucleic acid strands at a certain region (which is defined by primers and specific endonucleases in the reaction mixture) while at the same time synthesizing a new complementary strand.
A simple, isothermal strand displacement amplification process is described in EP-A 0 676 476. In this process only a single enzyme is needed, which is a polymerase. However, one needs at least 2 oligonucleotides per strand, i.e. altogether at least four oligonucleotides, for amplification.
A special form of PCR is nested-PCR (e.g. U.S. Pat. No. 5,340,728, EP-A 0 519 338, U.S. Pat. No. 4,683,195, U.S. Pat. No. 5,314,809).
Another special form of PCR is RT-PCR, wherein a RNA template is first transcribed into cDNA. One can use an enzyme which exhibits reverse transcriptase activity as well as DNA polymerase activity (e.g. U.S. Pat. No. 5,322,770).
Although all these methods have found their place in research (PCR must not be missed in a modern molecular biology laboratory) they are complex and are highly demanding so that they are hardly to be described as routine method. Also, there are high equipment--and reagent costs linked to these methods, since especially for thermocycling expensive high performance-thermostats are necessary (cf. R. Hoelzel, "Trends in Genetics", 6, 237-238 (1990); or U. Linz, Biotechniques, 9, 286-292 (1990), and on the other hand for the isothermal methods expensive enzymes have to be used.